Modern Pathology (2007) 20, 357–366 & 2007 USCAP, Inc All rights reserved 0893-3952/07 $30.00 www.modernpathology.org

Cellular and molecular mechanisms of abnormal following ischemia–reperfusion injury in human liver transplantation

Fariba Kalantari1,3, Dengshun Miao2, Anouk Emadali3, George N Tzimas3,*, David Goltzman2, Hojatollah Vali1, Eric Chevet1,2,3,4 and Patrick Auguste3,4,5

1Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada; 2Department of Medicine, McGill University, Montreal, QC, Canada; 3Department of Surgery, McGill University, Montreal, QC, Canada; 4INSERM, U889, Universite Bordeaux 2, Bordeaux, France and 5INSERM, U889, Universite Bordeaux 1, Talence, France

Recent studies suggest a possible link between calcification and ischemia–reperfusion injury following liver transplantation. Histological , immunolabeling, and biochemical and electron analyses were applied to assess the possible mechanism(s) of calcification in liver . Although light microscopy studies did not reveal the presence of large necrotic or apoptotic areas, electron microscopy showed the presence of membrane-bound vacuolar structures in hepatocytes, indicative of cell damage. Myofibroblasts were abundant in regions surrounding and within calcification. In these precalcified and calcified areas, myofibroblasts expressed bone-specific matrix proteins, such as osteopontin, type 1 collagen and bone sialoprotein. In addition, transforming growth factor beta (TGFb)-1 and BMP2, two growth factors implicated in osteoblast differentiation, and Runx2 and Msx2, two transcription factors targets of TGFb-1 and BMP2, were also expressed in these myofibroblasts. These data suggest that liver calcification following transplantation may be a consequence of precipitation of hydroxylapatite emanating from necrotic or apoptotic hepatocytes associated with proliferation of myofibroblasts expressing bone-specific matrix proteins. Modern Pathology (2007) 20, 357–366. doi:10.1038/modpathol.3800747

Keywords: liver calcification; ischemia-reperfusion; transplantation; myofibroblast

Cases of pathological liver calcification have been tal ischemia–reperfusion performed in pig liver,5 reported following a wide spectrum of injuries such thus suggesting a potential link between ischemia– as inflammatory states as well as primary benign or reperfusion and the observed . In spite malignant neoplasm.1 Furthermore, clinical reports of small patient number, calcification was found in have documented diffuse hepatic calcification de- 60% of liver disfunctions following human liver veloping after extensive ischemia2 or metastasis.3 In transplantation.6 all cases, calcification was a consequence of massive Ectopic calcification is characterized by the necrosis. In addition to necrosis, liver cell apoptosis pathological formation of hydroxyapatite crystals may also predispose to calcification as previously in nonskeletal tissues. Pathological calcifications described.4 Both necrosis and apoptosis have been can be subdivided into (i) ‘metastatic calcification’ shown to be massively triggered during experimen- occurring in undamaged tissues when and phosphate serum levels are elevated and (ii) ‘dys- trophic calcification’ occurring in injured tissue Correspondence: Dr P Auguste, PhD, GREF, INSERM, U889, Universite´, Bordeaux 2, 146 Rue Leo Saignat, Bordeaux 33076, when the levels of calcium and phosphate in serum 7 France. are normal. Dystrophic calcifications have many E-mail: [email protected]; features similar to physiological bone mineraliza- Professor H Vali, PhD. E-mail: [email protected]; tion such as the deposition of hydroxyapatite in Dr E Chevet, PhD. E-mail: [email protected] *Current address: Kypselis General Hospital, Athens, Greece. organic matrix containing type I collagen and Received 30 June 2006; revised 17 October 2006; accepted 19 noncollagenous proteins. Bone mineralization is October 2006 regulated by noncollagenous proteins present in Mechanisms of liver calcification F Kalantari et al 358 bone organic matrix such as osteopontin, bone Bovine Serum Albumin (BSA, Sigma) in PBS. The sialoprotein and osteocalcin.8 These bone-specific sections were then incubated with specific primary genes are activated during osteogenic differentiation antibodies for 1 h at 371C in blocking solution. by several growth factors such as transforming Mouse monoclonal antibodies against alpha smooth growth factor beta (TGFb)-1 and BMP2, which are muscle actin (a-SMA, clone 1A4, Dako, 1:1000), able to activate the specific transcription factors, Keratin 18 (Labvision Corporation, 1:500), human Msx2 and Runx2.9,10 Runx2 can cooperate with CD31 (Chemicon International, 1:1000), TGFb-1 TGFb- and BMP-specific Smads and stimulate (clone1D11, R&D Systems, 1:50) or rabbit sera transcription of the target genes required for osteo- against Runx2 (Santa Cruz, 1:50), Msx2 (Santa Cruz, genesis.11,12 1:50), BMP2 (Santa Cruz, 1:50) and Ki-67 (1:200) We previously reported two cases of graft calcifi- were used. After three washes in PBS, sections were cation following liver transplantation,6,13 in which incubated with Envision amplification system Dako patients had developed massive dystrophic liver (Cytomation Envision þ System labeled polymer- calcification after normal ischemia–reperfusion per- HRP anti-mouse or anti-Rabbit) for 30 min at room iods. In the present study, we have used a combined temperature and then washed three additional approach involving histological staining, immuno- times. Sections were stained with diaminobenzidine labeling, electron microscopy and biochemical (DAB, Dako) as chromogen and counterstained analysis to assess the involvement of the necrosis– with hematoxylin. Paraffin-embedded sections apoptosis and the osteogenesis in the process of (5 mm) were prepared for type I collagen (goat anti- liver calcification. human type I collagen antibody, Southern Biotech- nology Associates), osteocalcin (goat anti-mouse osteocalcin, Biomedical Technologies), osteopontin Materials and methods (rabbit anti-osteopontin, LF-123) and bone sialopro- Tissue Collection tein (rabbit anti-human bone sialoprotein, LF-6) detections following procedure described.17 After Calcified liver was obtained as previously described overnight incubation at room temperature, incuba- by Tzimas et al,13 macroscopically divided into tion with specific biotinylated rabbit anti-goat IgG or noncalcified, precalcified and calcified parts and goat anti-rabbit IgG (Sigma) and incubation with the snap frozen in less than 5 min. Three samples from Vectastain ABC-AP kit (Vector Laboratories, Inc., each region were dissected and used for biochemical Ontario, Canada), red pigmentation was produced and morphological studies. Control livers were by a 10- to 15-min treatment with Fast Red TR/ obtained from biopsies collected in the operating Naphthol AS-MX phosphate (Sigma; containing room in accordance with the McGill University 1 mM levamisole as endogenous alkaline phospha- Health Centre ethics regulations (ERB 05-003). tase inhibitor). After washing with distilled water, the sections were counterstained with methyl green and mounted with Kaiser’s glycerol jelly. In all experiments, the specificity of the staining was controlled by omitting the primary antibody. Hematoxylin and eosin staining and von Kossa staining were carried out following the procedure 14 15 described by Miao and Scutt and Xue et al, Cell Apoptosis Assay respectively. Masson–Goldner was performed as previously described.16 Hematox- Terminal-deoxynucleotidyl transferase-mediated ylin and eosin, von Kossa and Masson–Goldner nick-end labeling (TUNEL) staining of apoptotic trichrome were performed on paraffin-embedded cells was performed on 8 mm frozen sections accord- sections. For staining, 8 mm frozen ing to the manufacturer’s instruction (Roche) as sections were fixed with 10% formalin, partly described previously.18 decalcified in 10 mM ethylenediaminetetraacetic acid (EDTA, five washes of 2 min) and stained with 40 mM Alizarin red S (Sigma) for 5 min at room Transmission Electron Microscopy temperature. The sections were then washed three Approximately 0.02 cm3 tissue specimens were times with PBS and counterstained with hematox- fixed with 2.5% glutaraldehyde in 0.1 M cacodylate ylin (Vector Laboratories). buffer, washed with cacodylate buffer and post-fixed with 1% OsO4. The fixed material was then Immunohistochemistry dehydrated in graded concentrations of alcohol, and embedded in Epon resin. Ultrathin sections Frozen sections (8 mm) were fixed with 4% parafo- were prepared by ultramicrotomy using a diamond maldehyde in PBS (pH 7.4) and partly decalcified knife. To distinguish between mineralized and with 10 mM EDTA (five washes of 2 min). Sections nonmineralized cellular structures, lead citrate- were incubated in 3% hydrogen peroxide to quench and uranium acetate-stained and nonstained sec- endogenous peroxidases and blocked with 1% tions were imaged in transmission electron micro-

Modern Pathology (2007) 20, 357–366 Mechanisms of liver calcification F Kalantari et al 359 scopy using a JEOL JEM 2000FX transmission (osteopontin, bone sialoprotein and collagen I) and electron (Tokyo, Japan) operated at 1:1000 (TGFb-1). 80 kV. The chemical composition of the mineral phases was determined by energy dispersive spec- troscopy (Quartz XOne). Results Characterization of Liver Calcifications

Immunoblot Analyses Calcified livers were collected 15 days post-trans- plantation as previously described.13 All tissue Liver tissues (1–2 g) were homogenized in 1 ml of sections and proteins analyzed originated from PBS containing 1 Â protease inhibitor cocktail these livers. The presence of calcification within (Roche), 5 mM NaF, 1 mM EDTA and 1 mM Na3VO4 the liver tissues was evident by histochemical with an Ultra-Turrax T25. Tissue remnants were analysis using three different methods. Von Kossa removed by centrifugation at 1500 g for 20 min at staining showed the presence of phosphate as 41C. Membrane proteins were solubilized by addi- indicated black silver precipitation within liver tion of TX-100 to a 1% final concentration. After cells, most likely hepatocytes (Figure 1, top panels, incubation on ice for 1 h, insoluble material was von Kossa), whereas Alizarin Red S staining removed by centrifugation for 1 h at 100 000 g. indicated the presence of calcium (Figure 1, middle Protein concentrations were determined using the panels, Alizarin Red S). The co-existence of calcium Bradford reagent. Samples were immunoblotted as and phosphate suggests the precipitation of a previously described19 using antibody dilutions calcium phosphate phase within the calcified tissue. as follow: 1/200 (Runx2, Msx2 and BMP2), 1:500 Goldner–Masson trichrome staining (Figure 1,

Figure 1 Characterization of liver calcification. Von Kossa (top panels), alizarin red S (middle panels) and Goldner–Masson trichrome (bottom panels) staining of calcified, precalcified and noncalcified liver tissue sections. Top panels, black staining indicates the presence of phosphate precipitate. Middle panels, dark grey staining indicates the presence of Ca2 þ precipitate. Bottom panels, light grey staining indicates the presence of collagen. Magnification  20.

Modern Pathology (2007) 20, 357–366 Mechanisms of liver calcification F Kalantari et al 360 bottom panels, Goldner–Masson) confirmed the not associated to an organelle such as vacuole-like presence of collagen-containing matrix in the calci- or mitochondria (this study and that of Tzimas fied areas, as indicated by the light grey precipitates. et al6). Chemical analysis by energy-dispersive spectroscopy and high-resolution transmission elec- tron microscopy imaging indicated the presence of Tissue Necrosis and Apoptosis hydroxyapatite crystals similar to those observed in bone as well as hydroxyapatite formed in athero- Pathological calcifications have previously been sclerosis.22 Additional hydroxyapatite precipitates reported to be associated with necrosis and apopto- were also present in association with extracellular 4 sis. To assess the presence of necrotic areas in filamentous structures, suggesting a ‘classical’, ex- calcified liver, we first stained liver sections with tracellular matrix-associated calcification process hematoxylin and eosin. Unexpectedly, necrosis (Figure 3b). was not detected in any of the sections including noncalcified (Figure 2a), precalcified (Figure 2a) or calcified tissues (not shown). Similarly, TUNEL Identification of Major Cell Populations in Calcified staining showed labeling of only a small proportion Liver of cell nuclei (Figure 2b, arrows) but did not reveal massive apoptosis in any areas of the livers (Figure The presence of at least two major cell populations 2b). On the contrary, in precalcified region of the in precalcified areas was evident by hematoxylin liver, many cells were positively stained with the and eosin staining (Figure 2a). To further character- anti-Ki-67 antibodies, indicating their proliferative ize these cells, immunohistolabeling experiments state (Figure 2c, arrows). Although light microscopy were carried out on noncalcified and precalcified studies did not reveal massive necrosis/apoptosis, liver sections. Antibody against Keratin 18, a electron microscopy analysis of the precalcified specific marker of hepatocytes, showed a predomi- sections showed the presence of membrane-bound nant labeling in the parenchyma of noncalcified vacuolar structures, most likely altered mitochon- regions (Figure 4a; top panel). Interestingly, Keratin dria in hepatocytes (Figure 3c), indicative of cell 18-positive hepatocytes were not present in large damage.20,21 Furthermore, a significant number of numbers in the area adjacent to the calcified tissue intact hepatocytes within the calcified area showed where, at least, another cell type was predominant the presence of intracellular calcium phosphate (Figure 4a; bottom panel). Immunostaining with precipitates (Figure 3a and d), confirming the results anti-aSMA antibodies specific for myofibroblasts of von Kossa staining (Figure 1). Calcium phosphate and smooth muscle cells demonstrated the presence precipitates were found in the cytosol and were of significant numbers of immunostained cells at the

Figure 2 Characterization of necrotic, apoptotic and proliferative states. Hematoxylin and eosin staining (a), TUNEL staining (b) and Ki- 67 immunostaining (c) of liver sections obtained from noncalcified or calcified regions. Arrows show apoptotic nuclei (b) or cells in proliferation (c). Magnification  20 (a),  40 (b–c). In Ki-67 control experiment, no labeling was observed.

Modern Pathology (2007) 20, 357–366 Mechanisms of liver calcification F Kalantari et al 361

Figure 3 Electron microscopy analyses. Calcified (a) and fibrotic (b) regions were analyzed by transmission electron microscopy. The presence of dilated mitochondria was detected in numerous hepatocyte-like cells (c) as well as hydroxyapatite deposits within cell cytoplasms (d). Bars indicate either 5 mm(a–c), 1 mm(b)or2mm(d).

periphery of blood vessels in noncalcified regions ences were observed, however, in any of the sections (Figure 4b; top panel), indicating the presence of obtained from different regions (Figure 4b). These smooth muscle cells. Remarkably, a large number of results provide evidence for a massive proliferation cells at the edge of and within calcified areas (Figure of myofibroblasts in both precalcified and calcified 4b; bottom panel and not shown, respectively) also regions. expressed aSMA, suggesting the proliferation of myofibroblasts in these regions in response to liver 23 damage. Finally, as endothelial cell proliferation Expression of Specific Bone Markers in Calcified Liver has been reported to be associated with fibrosis,24 liver sections were labeled using an antibody against Physiological and is a CD31, a specific endothelium marker. No differ- highly regulated process involving the synthesis

Modern Pathology (2007) 20, 357–366 Mechanisms of liver calcification F Kalantari et al 362

Figure 4 Characterization of the different cell populations in noncalcified (top panels) and precalcified (bottom panels) regions. Immunostaining was performed with antibodies against the hepatocyte marker Keratin 18 (a), the myofibroblast marker aSMA (b) and the endothelial cell marker CD31 (c). Magnification  40. In control experiments, no labeling was observed; moreover, all three antibodies are mouse monoclonal from the same isotype and showed different labeling.

and secretion of collagenous and noncollagenous by immunoblot. These results confirmed the pre- proteins forming the bone matrix. To test whether sence of osseous extracellular matrix components bone matrix proteins played a role in liver calcifica- that may reflect an important fibrogenic process. tion, we first examined their presence in the They also suggest that an active bone-forming different regions of the calcified livers using im- process may operate simultaneously to myofibro- munolabeling using antibodies against type 1 col- blast proliferation. lagen and other noncollagenous proteins such as osteopontin, osteocalcin and bone sialoprotein. Osteopontin, a secreted glycoprotein with the ability TGFb Superfamily and Liver Calcification to bind to Ca2 þ was expressed in myofibroblasts in both calcified and precalcified areas (Figure 5a, The results presented above suggest a correlation osteopontin). In contrast, osteocalcin, a highly between the presence of collagenous and noncolla- abundant noncollagenous protein of bone matrix, genous bone-forming matrix proteins and myofibro- was only faintly expressed in myofibroblasts and blast proliferation associated with the deposition of hepatocytes in all three regions (data not shown). hydroxyapatite minerals. It is well established that The expression of type I collagen, the main organic the TGFb superfamily represents a common denomi- compartment of bone but also an extracellular nator to both fibrosis induction and bone formation. matrix component during fibrosis, was restricted to As a consequence, we analyzed the expression and myofibroblasts in both precalcified and calcified localization of two major members of the TGFb regions of the livers (Figure 5a, type 1 collagen). superfamily, namely TGFb-1 and BMP2 in noncal- Finally, bone sialoprotein, a promoter of osteoblast cified and in precalcified sections. Interestingly, differentiation and a potential nucleator of hydro- while TGFb-1 immunostaining labeled both myofi- xyapatite crystals, was expressed by both hepato- broblasts and hepatocytes (Figure 6a), BMP2 was cytes and myofibroblasts (Figure 5a, bone detected predominately in myofibroblasts and in sialoprotein). The results of immunohistochemistry rare cases in hepatocytes (Figure 6a). These results were confirmed by immunoblot analyses (Figure were confirmed by immunoblot analysis on proteins 5b). Indeed, the expression of both osteopontin extracted from the three regions of the liver. Indeed, and type I collagen increased in precalcified and both BMP2 and TGFb-1 protein expression levels calcified regions of the liver relative to noncalcified were increased in protein extracts from both areas. We were unable to detect bone sialoprotein precalcified and calcified regions (Figure 6b).

Modern Pathology (2007) 20, 357–366 Mechanisms of liver calcification F Kalantari et al 363

Figure 5 Bone matrix protein expression in the liver. Immunostaining (a) of calcified (top panels), precalcified (middle panels) and noncalcified (bottom panels) tissue sections was carried out using antibodies against osteopontin, or type 1 collagen or bone siaoloprotein. Magnification  20. In control experiments, no labeling was observed. In (b), Western blot analysis of calcified, precalcified and noncalcified tissue extracts were carried out using antibodies against osteopontin or type 1 collagen. Antibody against b- actin was used as a control.

To further confirm these observations and assess regions (Figure 6a), whereas in noncalcified areas their functional significance, we evaluated the only cells surrounding blood vessels were stained expression of specific transcription factors whose (Figure 6a). These results were confirmed by expression is regulated by TGFb-1 or BMP2 and immunoblot analysis, with enhanced expression of which have been implicated in osteoblast differen- Msx2 and Runx2 in protein extracts from precalci- tiation and calcified matrix synthesis. Two of these fied and calcified areas (Figure 6b). These data transcription factors Runx2 (Figure 6a) and Msx2 confirm the presence of TGFb-1 and BMP2 with (Figure 6a) were selected based on their previously their targets Runx2 and Msx2 in myofibroblasts. reported dependence on TGFb-1 and BMP2.25 These Although the overall calcification pattern in liver two transcription factors were mainly immunode- appears to be distinct, the expression of these tected in myofibroblasts present in precalcified proteins suggests that myofibroblasts may also

Modern Pathology (2007) 20, 357–366 Mechanisms of liver calcification F Kalantari et al 364

Figure 6 TGFb family growth factor and osteoblastic transcription factor expression. Immunostaining (a) of precalcified (right panels) and noncalcified (left panels) liver tissue sections was carried out using antibodies raised against TGFb-1, BMP2, Runx2 or Msx2. Magnification  40. In control experiments, no labeling was observed; moreover, BMP2, Runx2 and Msx2 are polyclonal rabbit antibodies and they showed distinct labeling patterns, especially in noncalcified sections. In (b), Western blot analysis of calcified, precalcified and noncalcified tissue extracts were carried out using antibodies against TGFb-1, BMP2, Msx2 or Runx2. Antibody against b-actin was used as a control.

undergo specific osteoblastic transformation leading transplanted livers and are attributed to ischemia– to the synthesis of calcified matrix similar to that reperfusion injury.2 On some occasions, however, observed in pathological aortic calcification.25 the development of macrocalcifications has been observed, leading to graft nonfunction and the requirement of a new transplantation.13 Unfortu- Discussion nately, the mechanism of pathological calcification is poorly understood, particularly in the case of liver Patients undergoing liver transplantation occasion- calcification. This study represents the first attempt ally develop complications such as calcifications to characterize the events leading to liver calcifica- which may in turn lead to graft dysfunction.13 tion obtained from partially calcified transplanted Frequently, microcalcifications are detected in livers.

Modern Pathology (2007) 20, 357–366 Mechanisms of liver calcification F Kalantari et al 365 Characterization of Liver Injury collagen I, four proteins found in calcified osseous matrix, leads to the assumption that activated The results of von Kossa, Alizarin red S staining, myofibroblasts may synthesize and secrete these transmission electron microscopy and energy-dis- proteins. Indeed, it has been shown that osteopontin persive spectroscopy analyses provide clear evi- produced locally by the smooth muscle cells dence that liver calcification is due to precipitation may serve as an inducible inhibitor of vascular of crystalline hydroxyapatite (Figure 1). Considering calcification.29 that the liver was removed 15 days after the initial Among the growth factors implicated in the transplant, no sign of liver necrosis was detectable mechanisms triggering myofibroblasts-dependent by hematoxylin and eosin staining. In contrast, calcification, TGFb-1 and BMP2 are strong osteo- transmission electron microscopy images showed inducers.30 Moreover, TGFb-1 is known to induce the presence of degenerating cells containing dilated type 1 collagen production in osteoblastic cells31 mitochondria, indicative of significant cell injury and to participate in type 1 collagen synthesis by (Figure 3c). It is possible that hematoxylin and eosin myofibroblasts and in calcium deposition in fibrotic staining may not be sufficient to completely identify areas. These growth factors were mainly expressed necrotic tissues. Furthermore, the paucity of TU- in myofibroblasts (Figure 6) of the liver samples NEL-positive cells at the periphery of calcified consistent with our findings on the production of regions suggested that massive apoptosis may not osseous matrix proteins by these cells. Msx29 and have occurred at the initial stage of liver calcifica- Runx210 are two transcription factors implicated in tion. In a pig model of liver ischemia–reperfusion, osteoblast differentiation and in arterial calcifica- sinusoidal cell and hepatocyte apoptosis has been tion. They are known targets of TGFb-1 and BMP2.25 shown to increase over time and to almost return 5 These transcription factors were highly expressed in to background level after 24 h of reperfusion. In myofibroblasts present in precalcified and calcified addition, more than 35% of hepatocytes have been 5 areas. This correlated with the expression of found to undergo necrosis. As our samples were osteopontin, osteocalcin, bone sialoprotein and collected 15 days post reperfusion, it cannot be type I collagen (this study and that of Otto et al10). excluded that a massive apoptosis occurred shortly Taken together, these data suggest that besides after transplantation and therefore may have been at the contribution of hydroxyapatite deposition the origin of the calcification process. A large within necrotic hepatocytes, myofibroblast-depen- number of Ki-67-positive proliferating hepatocytes dent calcification may also occur in the calcified was observed (Figure 2c), which suggested that a liver by a mechanism which is dependent on regeneration process may have been occurred after 26 TGFb-1 and BMP2 and their downstream transcrip- injury as has previously been reported. These data tional targets. are in agreement with the increased phosphoryla- In conclusion, in this study we have demonstrated tion of ERK1/2 in proteins extracted from the that liver macrocalcification following transplanta- precalcified and calcified regions (data not shown), tion may be the consequence of a generalized suggesting a growth factor-activated regeneration 27 response to injury involving osteoblast-like differ- process as previously described. In addition, the entiation of activated myofibroblasts. In addition, presence of abundant proliferating myofibroblasts we have demonstrated the presence of intracellular (aSMA-positive cells), as shown by Ki-67 staining accumulation of hydroxyapatite deposits in hepato- (Figure 2c), suggests the existence of mitogens in cytes, which may be associated with their ischemia/ their microenvironment. reperfusion-induced injury. We believe that these two mechanisms may both contribute to the process of liver calcification and its pathological Possible Mechanisms of Calcification consequences. Inhibition of at least one of these mechanisms may, therefore, prevent massive liver Histological Von Kossa and Alizarin red S staining calcification and improve organ recovery following suggested an intracellular accumulation of calcium injury. phosphate in damaged hepatocytes (Figure 1). Based on high-resolution transmission electron micro- scopy imaging and energy-dispersive spectroscopy Acknowledgements analysis, the structure and composition of the calcium phosphate is consistent with hydroxyapa- We thank Dr Kenneth Finnson, Division of Plastic tite. Morphological features characteristic for Surgery, McGill University, Montreal, Canada, for phagocytosis and endocytosis of hydroxyapatite providing anti-TGFb-1 antibody. This work was precipitates were not observed in both myofibro- supported by grants from the Canadian Institutes blasts and hepatocytes. Transmission electron for Health Research (CIHR) to EC and DG, National microscopy shows the presence of hydroxyapatite Science and Engineering Research Council (NSERC) within fibrotic regions similar to the pattern to HV. PA was supported by a sabbatical fellowship observed in arterial calcification.28 The detection from Universite´ Bordeaux 1 (Talence, France) and by of osteopontin, osteocalcin, bone sialoprotein and a grant from the Fondation pour la Recherche

Modern Pathology (2007) 20, 357–366 Mechanisms of liver calcification F Kalantari et al 366 Me´dicale (FRM). EC is a junior scholar from the yvitamin D3 play distinct and synergistic roles in Fonds de la Recherche en Sante´ du Que´bec (FRSQ). postnatal mineral ion homeostasis and skeletal devel- opment. Hum Mol Genet 2005;14:1515–1528. 16 van der Merwe SW, van den Bogaerde JB, Goosen C, et al. Hepatic osteodystrophy in rats results mainly References from portasystemic shunting. Gut 2003;52:580–585. 17 Miao D, Bai X, Panda D, et al. Osteomalacia in hyp 1 Stoupis C, Taylor HM, Paley MR, et al. The Rocky liver: mice is associated with abnormal phex expression and radiologic–pathologic correlation of calcified hepatic with altered bone matrix protein expression and masses. Radiographics 1998;18:675–685; quiz 726. deposition. Endocrinology 2001;142:926–939. 2 Shibuya A, Unuma T, Sugimoto T, et al. Diffuse hepatic 18 Miraux S, Lemiere S, Pineau R, et al. Inhibition of calcification as a sequela to shock liver. Gastroentero- FGF receptor activity in glioma implanted into the logy 1985;89:196–201. mouse brain using the tetracyclin-regulated expression 3 Shih WJ, Han JK, Magoun S, et al. Bone agent system. Angiogenesis 2004;7:105–113. localization in hepatic metastases. J Nucl Med Technol 19 Nguyen DT, Kebache S, Fazel A, et al. Nck-dependent 1999;27:38–40. activation of extracellular signal-regulated kinase-1 4 Kim KM. Apoptosis and calcification. Scanning Mi- and regulation of cell survival during endoplasmic crosc 1995;9:1137–1175; discussion 1175–1138. reticulum stress. Mol Biol Cell 2004;15:4248–4260. 5 Meguro M, Katsuramaki T, Kimura H, et al. Apoptosis 20 Lu Z, Dono K, Gotoh K, et al. Participation of and necrosis after warm ischemia–reperfusion injury autophagy in the degeneration process of rat hepato- of the pig liver and their inhibition by ONO-1714. cytes after transplantation following prolonged cold Transplantation 2003;75:703–710. preservation. Arch Histol Cytol 2005;68:71–80. 6 Tzimas GN, Afshar M, Emadali A, et al. Correlation of 21 Syntichaki P, Tavernarakis N. Death by necrosis. cell necrosis and tissue calcification with ischemia/ Uncontrollable catastrophe, or is there order behind reperfusion injury after liver transplantation. Trans- the chaos? EMBO Rep 2002;3:604–609. plant Proc 2004;36:1766–1768. 22 Watson KE. Pathophysiology of coronary calcification. 7 Anderson HC. Calcific diseases. A concept. Arch J Cardiovasc Risk 2000;7:93–97. Pathol Lab Med 1983;107:341–348. 23 Ramadori G, Saile B. Mesenchymal cells in the liver— 8 Donley GE, Fitzpatrick LA. Noncollagenous matrix one cell type or two? Liver 2002;22:283–294. proteins controlling mineralization; possible role in 24 Corpechot C, Barbu V, Wendum D, et al. Hypoxia- pathologic calcification of vascular tissue. Trends induced VEGF and collagen I expressions are asso- Cardiovasc Med 1998;8:199–206. ciated with angiogenesis and fibrogenesis in experi- 9 Davidson D. The function and evolution of Msx genes: mental . Hepatology 2002;35:1010–1021. pointers and paradoxes. Trends Genet 1995;11: 25 Shao JS, Cheng SL, Pingsterhaus JM, et al. Msx2 405–411. promotes cardiovascular calcification by activating 10 Otto F, Lubbert M, Stock M. Upstream and downstream paracrine Wnt signals. J Clin Invest 2005;115: targets of RUNX proteins. J Cell Biochem 2003;89: 1210–1220. 9–18. 26 Rozga J. Hepatocyte proliferation in health and in liver 11 Hanai J, Chen LF, Kanno T, et al. Interaction and failure. Med Sci Monit 2002;8:RA32–RA38. functional cooperation of PEBP2/CBF with Smads. 27 Rosario M, Birchmeier W. How to make tubes: Synergistic induction of the immunoglobulin signaling by the Met receptor tyrosine kinase. Trends germline Calpha promoter. J Biol Chem 1999;274: Cell Biol 2003;13:328–335. 31577–31582. 28 Giachelli CM. Vascular calcification mechanisms. J Am 12 Miyazono K, Maeda S, Imamura T. Coordinate regula- Soc Nephrol 2004;15:2959–2964. tion of cell growth and differentiation by TGF-beta 29 Speer MY, Chien YC, Quan M, et al. Smooth muscle superfamily and Runx proteins. Oncogene 2004;23: cells deficient in osteopontin have enhanced suscept- 4232–4237. ibility to calcification in vitro. Cardiovasc Res 2005;66: 13 Tzimas GN, Afshar M, Chevet E, et al. Graft calcifica- 324–333. tions and dysfunction following liver transplantation. 30 Wan M, Cao X. BMP signaling in skeletal development. BMC Surg 2004;4:9. Biochem Biophys Res Commun 2005;328:651–657. 14 Miao D, Scutt A. Histochemical localization of alkaline 31 Palcy S, Goltzman D. Protein kinase signalling path- phosphatase activity in decalcified bone and cartilage. ways involved in the up-regulation of the rat alpha1(I) J Histochem Cytochem 2002;50:333–340. collagen gene by transforming growth factor beta1 and 15 Xue Y, Karaplis AC, Hendy GN, et al. Genetic models bone morphogenetic protein 2 in osteoblastic cells. show that and 1,25-dihydrox- Biochem J 1999;343(Part 1):21–27.

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